The Interaction Of Light And Biological Molecules Biology Essay

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The experimental objective was to determine biological molecules and the concentration of solutions through the utilization of a spectrophotometer, fluorometer and Beer's Law. The concentration of a solution of NADH was determined by direct application of Beer's Law at 340 nm, with a - 24% error. The concentration of solutions of proteins were determined by the Bradford assay, against bovine γ-globulin standards, with a + 10.4 % error for a bovine γ-globulin unknown and a + 45% error for the protein content of Milk A and B. The concentration of a solution of Ponceau S in an artificial urine matrix was determined by standard addition, with an error of - 35 %. The identity of a sample of olive oil A1 and 1B were determined qualitatively by fluorescence emission.

Introduction

The spectrophotometer passes light through solution, housed within a cuvette. The amount of light absorbed can then be determined through the mathematical equation A = εbc, known as Beer's Law (Manz et al 2004). The terms in the equation, A = εbc, represents absorbance, A, molar absorptivity, ε, path length through the solution and concentration of light through absorbing molecule, c (Boyer, 2006). When the absorptivity is known, Beer's Law can then be utilized (Manz et al 2004). The concentration of NADH unknown was determined by direct application of Beer's Law, with the spectrophotometer set at 340nm (Boyer, 2006).

In cases when molar absorptivity is unknown, a standard curve is useful for determining the concentration of the absorbing unknown (Boyer, 2006). Beer's Law equation is rewritten as A=kc. The variable k represents k=εb (Boyer, 2006). A straight line is yielded when absorbance is plotted versus concentration of known solution. The trendline equation acquired, from the standard curve, can calculate the unknown solution.

The Bradford protein assay is employed in this experiment since proteins have no useable wavelengths of absorbance ( Boyer, 2006). Bradford assay allows proteins wavelength to be usable because of the Coomassie Brilliant Blue G dye is negatively charged (Boyer, 2006). The reaction between the negatively charged dye and positive protein changes the dye from red to blue (Boyer, 2006). When the reaction occurs, the protein wavelength of absorbance is usable at maximum absorbance of 595nm (Boyer, 2006).

A fluorometer is utilized in this experiment. The fluorometer measures the longer emitted wavelengths of fluorescence. Emitted light from biological molecules, such as chlorophyll, is known as fluorescence (Wilson et al 2010). The fluorometer measures the excited electrons of molecules that have absorbed a photon of light (Wilson et al 2010). The amount of emitted light is directly proportional to the quantity of fluorescing samples; this allows Beer's Law to be applied for quantitative analysis of fluorescing molecules (Boyer, 2006).

Materials and Methods

In Part A of this experiment, a blank cuvette filled with distilled water, was utilized to zero the Spectronic 1201 spectrophotometer set at 340 nm (Boyer, 2006). The concentration of the unknown, number 3, NADH solution was determined by direct application of Beer's Law (Boyer, 2006).

In Part B, the protein dye reagent was utilized to determine the concentration of a protein solution of an unknown concentration. In table 2, a standard curve was acquired by pipetting 20uL of standard bovine y-globulin solution to 1.00mL of Bradford protein assay (Boyer, 2006). The unknown protein P5 and two milk samples, previously diluted 1:50 in PBS buffer, of unknown M4 and M6 were dealt in the same method (Boyer, 2006). After the solutions were transferred to cuvettes, an inverted three times, the solutions sat at room temperature for twenty minutes (Boyer, 2006). At the twenty minute marker, a Spectronic 601 spectrophotometer, set at 595 nm, was employed to determine the absorbance of the solutions against the Bradford reagent cuvette blank (Boyer, 2006).

In Part C, standard addition was utilized to determine the concentration of Ponceau S dye, a red solution, in artificial urine matrix, a yellow solution (Boyer, 2006). In Table 3, a standard curve was acquired by pietting of 1 M Ponceau standard solution to 2.00 mL urine sample in cuvettes two through six (Boyer, 2006). A Spectronic 601 spectrophotometer, set at 595 nm, was employed to determine the absorbance of the solutions against cuvette water blank (Boyer, 2006).

The Vernier Spectro Vis Plus in fluorometer mode was employed in Part D in order to identify the fluorescence of two unknown olive oil samples, 1A and 1B. The fluorometer, set at 405 nm, to measure olive oil samples extra virgin, classic and extra light to identify the grade of the unknown samples (Boyer, 2006).

Results

Data

Part A - Determination of NADH concentration by direct application of Beer's Law

Table 1

NADH unknown number

N 03

Absorbance at 340 nm of unknown NADH solution

0.592

Part B - Determination of protein concentration by standard curve

Samples: Bovine γ-globulin protein unknown UP5, reduced-fat chocolate milk A, reduced-fat milk B

Table 2

Tube Number

Contents

Protein Concentration, mg/mL

Absorbance

blank

PBS buffer

0.000

0.000

1

Bovine γ-globulin

0.125

0.119

2

Bovine γ-globulin

0.250

0.265

3

Bovine γ-globulin

0.500

0.489

4

Bovine γ-globulin

0.750

0.786

5

Bovine γ-globulin

1.000

0.994

6

Bovine γ-globulin

1.500

1.495

7

Bovine γ-globulin

2.000

1.899

8

Bovine γ-globulin unknown UP5

unknown

1.350

9

Reduced-fat milk, 1:50 dilution A

unknown

0.950

10

Reduced-fat chocolate milk, 1:50 dilution B

unknown

1.899

Part C - Determination of Ponceau S by Standard Addition

Sample: Artificial urine matrix unknown # 2

Table 3

1 M Ponceau S standard, μL, added

μmoles Ponceau S added

Absorbance

0

0

0.050

2

2

0.082

3

3

0.104

4

4

0.125

5

5

0.178

Part D - Identification of Olive Oil Sample by fluorometry

Olive Oil Samples: Extra Virgin, Classic, Extra Light, Unknown Set 1A and 1B

Table 4

Sample

Fluorescence at 405 nm

Extra Virgin

0.4

Classic

0.1

Extra Light

0.0

1A

0.4

1B

0.1

Calculations

Part A - NADH Determination

Determination of NADH concentration in unknown N 3

A = ε340bC

0.592 = 6220 cm-1M-1 X 1 cm X C

C = 9.52x 10-5 M X 1000 = 0.095mM

Determination of error in NADH determination

% error = [experimental value - actual value]/actual value x 100%

= [0.095 mM - 0.125 mM]/0.125 mM x 100%

= - 24%

Part B - Protein Determination by Bradford Assay

Determination of the standard curve

Trendline: Absorbance = 0.962 mg protein/mL + 0.0194

Determination of unknown concentrations utilizing trendline equation.

Determination of protein in bovine γ-globulin UP 5:

Absorbance = 0.962 mg protein/mL + 0.0194

1.350 = 0.962 mg protein/mL + 0.0194

mg protein/mL = 1.38 mg protein/mL

Determination of error in UP 5determination

% error = [experimental value - actual value]/actual value x 100%

= [1.38 mg/mL - 1.25 mg/mL]/1.25 mg/mL x 100%

= + 10.4 % error

Determination of protein concentration in milk samples

Milk A: Lactaid

Absorbance = 0.962 mg protein/mL + 0.0194

0.950 = 0.962 mg protein/mL + 0.0194

mg protein/mL of dil. milk sample = 0.967 mg/mL dil. milk

mg protein/mL of undil. sample = 0.967 mg/mL dil.milk x 50 mL dil.milk/1 mL undil. milk

mg protein/mL of undiluted sample = 48.35 mg/mL = 11.6 g/240 mL

Determination of error milk A

% error = [experimental value - actual value]/actual value x 100%

= [11.6 g/240 mL - 8 g/240 mL]/8 g/240 mL x 100%

= + 45% error

Milk B: Evaporated milk

Absorbance = 0.962 mg protein/mL + 0.0194

1.899 = 0.962 mg protein/mL + 0.0194

mg protein/mL of dil. milk sample = 1.95 mg/mL dil. milk

mg protein/mL of undil. sample = 1.95 mg/mL dil.milk x 50 mL dil.milk/1 mL undil. milk

mg protein/mL of undiluted sample = 97.5 mg/mL = 2.9 g/30 mL

Determination of error milk B

% error = [experimental value - actual value]/actual value x 100%

= [2.9 g/30 mL - 2 g/30 mL]/2 g/30 mL x 100%

= +45% error

Part C - Ponceau S Determination by Standard Addition

Determination of Standard Curve

Trendline: Absorbance = 0.0241 umoles of Ponceau S added + 0.0403

Determination of umoles of Ponceau S in urine #2

Absorbance = 0.0241 μmoles of Ponceau S added + 0.0403

0 = 0.0241 μmoles of Ponceau S added + 0.0403

μmoles of Ponceau S = -1.67 μmoles.

The amount -1.67 must be removed from the original sample, to have an absorbance equal to 0.

Therefore, the concentration of Ponceau S in urine #2 is 1.67 μmoles/2.00 mL = 0.835 μmoles/mL

Determination of error in Ponceau S determination:

% error = [experimental value - actual value]/actual value x 100%

= [0.835 μmoles/mL - 1.3 μmoles/mL]/ 1.3 μmoles/mL x 100%

= - 35 % error

Results

The unknown #3 of NADH concentration was determined to be 0.095mM; the percent error from the actual value of 0.125 mM is - 24%. The unknown concentration of protein UP5 was determined to be 1.38 mg/mL; the percent error from the actual value of 1.25 μg/μL is + 10.4 %. The concentration of protein in milk A, lactaid, was determined to be 11.6 g/240 mL; the percent error from the label value of 8 g/240 mL is + 45%. The concentration of protein in milk B, Evaporated, is determined to be 2.9 g/30 mL; the percent error from the label value of 2 g/30 mL is +45%. The concentration of Ponceau S in artificial urine sample #2 is determined to be 0.835 umoles/mL; the percent error from the actual value is - 35 %. Olive oil sample 1A was extra virgin and olive oil sample 1B was classic.

Discussion

Part A of the experiment, the concentration of NADH is derived directly from the absorbance of the solution and Beer's law (Boyer, 2006). NADH concentration could have been affected from poor technique with a micropipette or damage cuvette. If the micropipette was not calibrated correctly the wrong amount of solution would be transferred into each cuvette. . The cuvette could have received the correct amount of the solution but a scratch on the cuvette or labeling the cuvette on the wrong side could have also affected NADH concentration. Degradation of the NADH could result in an experimental concentration lower than the actual value, if the filled cuvette was exposed to light for any length of time before the absorbance was determined.

In Part B, concentration of protein in the bovine γ-globulin unknown depends upon the linearity of the standard curve (Boyer, 2006). The linearity of the standard curve was acceptable, as demonstrated by the R2 value of 0.9981; this depends in upon the accuracy of pipetting. The agreement of the experimental determination of the bovine γ-globulin unknown with its actual value acts as a control for the experiment. The extremely large deviations of the milk results from the expected values can be attributed to two causes. Errors that could have accrued in Part B of this experiment are poor technique with a micropipette or cuvette. In addition, milk contains many more substances beside proteins which can interfere with the absorbance measurements.

In Part C, the concentration of Ponceau S artificial urine was determined by the standard curve (Boyer, 2006). The linearity of the standard curve is acceptable, as demonstrated by the R2 value of 0.9304; this depends upon the accuracy of pipetting. On the Ponceau S graph most values are not on the linear line; the values are close enough to give reasonable data but the graph shows pipetting technique requires improvement.

In Part D, Emitted fluorescence from the olive oil samples identified the unknowns A1 and B1from the corresponding values. Malfunction of the Vernier Spectro Vis Plus or reading the data incorrectly can change the data collected.

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